RU2370280C2 - METHOD OF HER2/neu ONCOGENE ANTIBODY SUPERPRODUCTION IN PLANT - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention claims method of antibody production in plant cell for specific binding of HER2/neu oncoprotein, involving: (a) obtainment of one or more expression vectors guiding synthesis of heavy or light strand in plant cell for antibody or its antigene-binding fragments (dsFv, scFv, scFv-Fc); (b) insertion of given one or more vectors into plant cell; (c) plant cell cultivation in suitable conditions for combined expression of the vectors in cell. Invention discloses plant cell producing antibody for interaction with HER2/neu oncoprotein and transformed by one or more expression vectors guiding synthesis of heavy or light strand of antibody or its antigene-binding fragments (dsFv, scFv, scFv-Fc).

EFFECT: application in breast cancer diagnostics and treatment.

27 cl, 13 dwg, 4 ex

Description

Technical field

The invention relates to biotechnology, genetic engineering and medicine and can be used to create plants - superproducers of antibodies, in particular against HER2 / neu-positive cancer cells. Such antibodies can be used to diagnose and treat breast cancer.

State of the art

Breast cancer now affects every ninth woman, and its spread is increasing every year. Early treatment is a surgical intervention. However, the insidiousness and danger of breast cancer consists in rapid metastasis, and then chemotherapy and radiotherapy are prescribed. Several years ago, trastuzumab, created by Genentech (Genentech, USA) and manufactured by Hoffman La Roche (Hoffman La Roche, Switzerland), was introduced into medical practice. This medicine is based on humanized monoclonal antibodies (MA) (huMab4D5-8 or rhuMabHer2) produced by the ATCC CRL-10463 mouse hybridoma from a collection of US microorganism strains and cell cultures. These widespread antibodies are just one type of known antibody that interacts with the HER2 oncoprotein (from Human Epidermal growth factor Receptor 2). The HER2 / neu oncogene is an ErbB2 transmembrane tyrosine protein kinase with a molecular weight of 185 kDa. Over 30% of breast cancer patients suffering from the so-called HER2 / neu-positive and most dangerous form of cancer have superproduction of this oncoprotein, which causes accelerated metastasis and resistance to therapeutic effects. An antibody introduced into the patient’s bloodstream interacts with the extracellular part of HER2 / neu and inhibits the division of cancer cells, rarely accompanied by the destruction of cancer cells. In combination with chemotherapy, huMab4D5-8 antibodies have a pronounced therapeutic effect. Genentec first filed its application for the use of MA against SK-BR-3 breast cancer cells overexpressing the Her2 / neu oncogene on August 5, 1994 and received patent number 5,677,171 on October 14, 1997 (inventors Hudziak RM, Shepard N.M., Ullrich A., Fendly V.M.). Summary of the invention Genentec declares (a) the creation of MA against the Her2 / neu receptor, inhibiting the growth of tumor cells; (b) MA binding the extracellular domain of Her2 / neu and sensitizing cells to the action of the cytotoxic factor. Subsequently, during the first half of 1998, applications were submitted for the use of huMab4D5-8 in a clinic (US patent 6,165,464, WO9704801, WO9817797). A little later, the use of MA 454C11 produced by the HB 8484 hybridoma was proposed for the same purpose by Chiron (Chiron Corp., USA) (application dated June 7, 1995), and it was granted US patent 6,054,561 on April 25, 2000.

In Russia, the humanized MA huMab4D5-8, known as trastuzumab or under the brand name “Herceptin,” is successfully used. It has been shown that the use of herceptin reduces the risk of developing distant metastases and increases the life expectancy of patients (Ganshina and Lichinitser. 2005. Farmateka No. 18, 8-11). The cost of treating Her2 / neu-positive breast cancer with this drug is very high at $ 70,000 [Fleck L. "The costs of caring: Who pays? Who profits? Who panders?" Hastings Cent Rep 36 (3): 13-17]. In general, the cost of production of any target protein in animal cell culture is quite high and exceeds $ 10,000 per gram of protein (Danielli et al., 2001. TRENDS in Plant Science 6, 219-226). This is several times higher than the cost of producing a similar protein in yeast, insect cells, and even more so in bacteria. Typically, MAs are obtained using an animal cell culture, Chinese hamster ovary (CHO) ovary cells, and mouse myeloma cells. The level of MA production is currently quite high and can reach 3 g MA per liter of animal cell culture (Zhang et al., 2006. Biotechnol Bioeng. 95, 1188-1197). However, its high cost consists of the costs of equipment, a maintenance and sterility system, as well as a certification and control system for environments that use animal products and increase the risk of MA contamination with prions and viruses.

Plants have several advantages over bacterial, yeast, and mammalian cells for the production of foreign proteins. Plants as “factories” (a) do not contain viruses and prions pathogenic for humans, and (b) do not require the use of expensive equipment (fermenters), culture media, and a sterility system. The cost of growing the original experimental plants is incomparably lower than the cost of culturing bacterial, yeast, or animal cells. The technology of “temporary” production (transient expression) of proteins in a plant allows producing a foreign protein in an amount reaching 10-30% of the total soluble protein of a plant in a short time (5-10 days), when the target gene is included in a binary vector with subsequent delivery into the cells of an infected plant by agroinoculation or agroinfiltration (Kapila et al., 1997. Plant Science 122, 101-108). Under these conditions, the level of accumulation of the target protein is determined by the efficiency of (a) transcription of the binary vector (Rombauts et al., 2003. Plant Physiol. 132, 1162-1176), (b) replication (Asurmendi et al., 2004. Proc. Natl. Acad Sci. USA 101, 1415-1420; Marrillonet et al., 2005. Nature Biotechn. 23, 718-723), (c) protecting the transcript from degradation (Voinnet et al, 2003. Plant J.33, 949-956) , (g) the stability of the target protein.

There are two reports in the literature on the production of a complete anti-cancer antibody in a plant. In transgenic tobacco plants, a small amount (0.02% of soluble proteins) of antibodies against colorectal cancer was accumulated (Co. et al., 2006, Proc. Natl. Acad. Sci. USA 102, 7026-7030). The use of viral vectors and a transient (transient) expression system allowed the accumulation of up to 3-5% of the soluble protein of anti-cancer antibodies A5 (Giritch et al., Proc.Natl. Acad. Sci. USA 103, 14701-14706). However, so far no one has been able to express an anti-HER2 / neu oncogen antibody in the plant.

Disclosure of invention

The present invention relates to a method for producing an antibody in a plant cell that specifically binds HER2 / neu oncoprotein. The method of the present invention includes:

(a) providing one or more expression vectors directing in the plant cell the synthesis of the heavy, light chain of an antibody or its antigen-binding fragments (dsFv, scFv, scFv-Fc);

(b) introducing said one or more vectors into a plant cell;

(c) cultivating a plant cell under conditions providing for co-expression of said vectors in the cell.

In one embodiment, the plant cell is an isolated cell or is in a plant cell culture, plant tissue culture, plant organ culture, the whole plant or part thereof.

In one embodiment, the method comprises the use of one or more expression vectors that enable the synthesis of non-amplifying RNA. In one preferred embodiment, said one or more expression vectors enable the synthesis of non-amplifying RNA under the control of an inducible promoter.

In an alternative embodiment, the method comprises the use of one or more expression vectors, which are viral expression vectors. In one preferred embodiment, the viral expression vectors used are vectors based on the genome of a DNA-containing virus. In yet another preferred embodiment, said viral expression vectors are vectors based on the genome of an RNA-containing virus. Most preferably, the viral expression vectors based on the genome of a DNA or RNA-containing virus provide for the synthesis of viral RNA using an inducible promoter. Preferably, the viral expression vectors are selected so as to exclude competition from each other in the same cell.

In one of the preferred embodiments, the expression vector provides the synthesis of tobacco mosaic virus RNA encoding an antibody heavy chain. In yet another preferred embodiment, the expression vector provides the synthesis of tobacco mosaic virus RNA encoding an antibody light chain.

In a further preferred embodiment, the expression vector provides synthesis of potato X virus RNA encoding an antibody heavy chain. In yet another preferred embodiment, the expression vector provides for the synthesis of potato X virus RNA encoding an antibody light chain.

In yet another embodiment, the expression vectors provide synthesis of the light and heavy chains of an antibody in the same cell.

In a further embodiment, the produced antibody comprises a signal peptide capable of addressing the antibody to the cell compartment or secretion outside the cell.

In one embodiment, the antibody belongs to an immunoglobulin of class G or A, or M, or D, or E.

In yet another embodiment, the plant cell is a cell of a dicotyledonous or monocotyledonous plant. In a preferred embodiment, the dicotyledonous plant belongs to the nightshade family. Even more preferably, when the plant in the nightshade family is a plant of the genus Nicotiana. Most preferably, a plant of the genus Nicotiana is N. tabacum or N. benthamiana.

In yet another embodiment, the dicotyledonous plant is a plant of the Brassicacea, Legume, or Chenopodiaceae families.

In a further embodiment, expression vectors are introduced into the plant cell by agroinjection.

In yet another embodiment, the expression vectors provide a stable transformation. In an alternative embodiment, the expression vectors provide transient transformation.

In a further aspect, the present invention relates to a plant cell that produces an antibody that interacts with the HER2 / neu oncoprotein. In one embodiment, the cell is stably transformed with a genetic construct directing antibody synthesis. In an alternative embodiment, the plant cell is transiently transformed with a genetic construct directing antibody synthesis.

Brief Description of the Figures

Figure 1. The structure diagram of the binary vector pA11866 encoding the light (L) chain of the HER2 / neu-specific antibody, where RB and LB are the right and left parts of the site of embedding the binary vector in the plant genome, respectively; 35S — transcriptional promoter of cauliflower mosaic virus (VMCC); PolyA / T - VCCA polyadenylation signal / terminator for transcription of VCCA genomic RNA. S1 leader - 5'-untranslated region of the genomic RNA of WCC.

Figure 2. The accumulation of the light chain (L-chain) in the leaves of plants agroinjected with the vector pA11866 described in FIG. The leaves of the N. benthamiana plant were agroinjected with pA11866 in the presence of a binary vector encoding the suppressor of silencing p19 of the bushy tomato dwarf tombus virus virus (lane 1), or the pA11691 vector directing the synthesis of artificial tRNA in the cell under the control of the tRNA promoter (lane 2). Proteins were analyzed in Western blotting using antibodies specific for the Her2 / neu-specific human antibody light chain.

Figure 3. The structure diagram of the binary vector pA11903 encoding the heavy (H) chain of the Her2 / neu-specific antibody. Symbols are the same as in Figure 1.

Figure 4. Accumulation of Her2 / neu-specific antibodies in a plant agro-injected with non-amplifying binary vectors pA11866 and pA11903, and their purification on a protein G-sepharose column.

Figure 5. Structural diagram of the binary vector pA11520 based on the crvTM cDNA and encoding the heavy (H) chain of the Her2 / neu-specific antibody, where (from left to right) LB is the left part of the site of the integration of the binary vector into the plant genome; T - nopaline synthase terminator; NPT - neomycin phosphotransferase gene; P - nopalinsynthase transcriptional promoter; Act2 - actin transcriptional promoter 2 from arabidopsis; krVTM replicase gene; TB - transport protein krVTM, H-chain - heavy chain; NTR - 3 'untranslated region of the genomic RNA of krVTM; T - nopaline synthase terminator; RB is the right part of the site of embedding a binary vector in the plant genome. Arrows indicate the direction of transcription.

6. Structural diagram of the binary vector pA11333 based on PVA cDNA and encoding the light (L) chain of a Her2 / neu-specific antibody, where (from left to right) LB is the left part of the site of insertion of a binary vector into the plant genome; T - nopaline synthase terminator; NPT - neomycin phosphotransferase gene; P - nopalinsynthase transcriptional promoter; 35S - transcriptional promoter of WCC; PVX RdRp-replicase PVX, L-chain - light chain; NTR - 3 'untranslated region of genomic RNA of PVX; T - 35S terminator VMTSK; RB is the right part of the site of embedding a binary vector in the plant genome. Arrows indicate the direction of transcription.

7. Accumulation of Her2 / neu-specific antibodies in a plant agroinjected with the binary vectors pA11520 and pA11333. Figa. Plant protein extracts 3 (lane 1) and 7 (lane 2) days after agroinjection with the pA11520 and pA11333 vectors were fractionated by electrophoresis, the gels were stained with Coomassie. Figb. Detection of a Her2 / neu-specific antibody by Western blotting with antibodies to the alpha and kappa chains of human antibodies. Arrows indicate: Rubisco is a heavy subunit of chloroplast ribulose bisphosphate carboxylase.

Fig. 8. Accumulation of Her2 / neu-specific antibodies in a plant agroinoculated with the viral binary vectors pA11520 and pA11333 and their purification on a column with protein G-sepharose. Protein zones corresponding to the heavy (H) and light chain (L) antibodies are noted.

Fig.9. Antibodies isolated from plants react with the extracellular part of the Her2 / neu oncogen. Figa. - Cytological analysis of SKBR-3 cells expressing the Her2 / neu oncogen and treated with antibodies isolated from plants. Binding specificity was tested using rabbit antibodies conjugated to horseradish peroxidase and reacting with human antibodies. Figb. - SKBR-3 cells after treatment with diagnostic antibodies that specifically react with Dako Her2 / neu (positive control). Figv and Figg - Negative controls, respectively, to A and B, obtained by treating SKBR-3 cells only with rabbit antibodies.

Embodiments of the invention

The basis of the present invention is the fact discovered by the author that genes encoding the heavy and light chains of MA against the HER2 / neu oncogen can be expressed in a plant cell to produce a fully functional antibody that specifically binds to “own” antigen. The proposed method involves the creation of expression vectors that direct the synthesis of a heavy (H) and light (L) chain of an antibody in a plant cell, the introduction of these vectors into a plant cell, and the cultivation of a plant cell under conditions that co-express these vectors in the cell. In general, the proposed method can be used to express any anti-cancer antibodies whose amino acid sequence is known and published, for example, bevacizumab (avastin) (Kohen et al., 2007. Oncologist 12, 713-718; US patent 7,241,444), panitumumab (Easley and Kirkpatrick , 2006. Nature Review 5, 987-988; US Patent Application No. 20070179086). Currently, a number of MAs are known against the extracellular domain of the HER2 / neu oncogen, which can be used to treat breast cancer, and the genes encoding them. In particular, genes encoding antibodies against the extracellular domain of the HER2 / neu oncogen can be isolated from hybridoma ICO85 synthesizing murine MA directed against the HER2 / neu oncogen (see Example 1). mRNA encoding the antibody light and heavy chains can be formed both from replicating vectors created on the basis of viral genomes (see Example 3), and from non-amplifying mono (see Example 2) or polycistronic mRNAs using the ribosome internal landing site ( Dorokhov et al., 2002. Proc. Natl. Acad. Sci. USA 99, 5301-5306). In the context of the present invention, expression vectors encoding the light and heavy chains of the anti-HER2 / neu oncogen antibody can be generated based on the Bin19 binary vector (see FIGS. 1 and 3 of Example 2). These vectors provide synthesis and accumulation in the plant of antibodies in an amount sufficient for isolation and purification (see Figure 4 of Example 2).

An advantage of the present invention is that it enables the production of not only full-sized antibodies in the plant, but also antigen-binding fragments thereof, for example dsFv, scFv, scFv-Fc, as well as camel antibodies that contain only heavy chains (see Jain et al ., 2007. Trends in Biotechnology 25, 307-316).

Simultaneous expression of antibody chains in a plant cell using viral vectors is possible provided there is no competition between viruses. Our experiments show that TMV and CVC not only do not compete with each other, but CVC even stimulates the reproduction of TMV. The accumulation of the heavy chain from the TMV vector increases in the presence of PVI encoding the antibody light chain (see Example 3). An analysis of the mechanisms of reproduction of syndbis-like phytoviruses shows that they compete at the stage of formation of the so-called viral “factories”, when viral replicase interacts (attaches) with the membranes of the endoplasmic reticulum. TMV and CVC do not compete with each other at the stage of formation of the replicative complex and the viral “factory”. The turnip yellow mosaic virus (HMT), which forms the viral “factory” on chloroplast membranes, also does not compete with TMV and CVC and therefore can be used to synthesize antibody chains in a single cell. This approach can be used to select other non-competing viral pairs.

The present invention provides for the use of non-viral vectors for the synthesis of antibodies in a plant cell. These vectors do not compete with each other in the cytoplasm. However, their synthesis and stability in the cytoplasm is controlled by the mechanism of silencing (silencing) of the genes, which can lead to a decrease in the level of production of the target antibody. In the event that there is indeed a decrease in the production of the target protein as a result of gene silencing, the present invention provides for the use of antisilencing proteins. As an example of an anti-silencing protein, for example, the P19 protein of the bushy dwarf virus of tomatoes, the gene of which is easy to synthesize, can be mentioned (see example 2). The same anti-silencing effect can have short non-coding RNAs synthesized under the control of RNA polymerase III (see Figure 2).

The plant cell expressing the antibody may be in the form of an isolated cell devoid of a cell wall (protoplast), or as part of a cultured explant (tissue, organ) or the whole plant. Cultivation of cells under artificial conditions occurs in a sterile medium of known composition and under controlled conditions at a constant temperature (26-28 ° C) and light. The present invention contemplates the possibility of using non-coding RNAs to stimulate the production of antibodies in cultured cells, both in the form of a suspension culture and in plant explants cultivated on a solid medium, or in a whole plant grown in a suitable medium, such as a natural substrate (e.g., soil, soil substitutes), or in hydroponic culture. An antibody can accumulate inside a plant cell or, by introducing a signal sequence into it, can be exported to the culture medium. Antibody extraction can occur from intracellular contents by destroying the cells of the culture or directly from the culture medium when secretion into the culture medium is achieved by incorporating the signal sequence into the antibody.

DNA encoding antibody chains can be introduced into a plant cell by methods known to those skilled in the art, for example, by electroporation, microprojectile bombardment, microinjection, and virus infection. In addition, DNA can be delivered to a plant cell using Agrobacterium tumefaciens. For transient expression of a foreign gene in leaf tissue, the method of agroinfiltration and agroinoculation is more often used (Kapila et al., 1997. Plant Science 122, 101-108). The transformation of plant cells can be stable, accompanied by the integration of the introduced DNA into the cellular genome. The desired effect can also be achieved by introducing DNA into the nucleus and transient (temporary) RNA synthesis without stable integration of DNA into the host genome (see Examples 2 and 3).

An important and decisive advantage of the present invention is that an anti-HER2 / neu antibody synthesis system in a plant can achieve a high level of cheap protein production. Our experiments show that from 1 kg of leaves of N. benthamiana up to 300-500 mg of purified antibody can be isolated (3-5% of soluble protein). Using chromatography on a protein G or A column, a pure anti-Her2 / neu oncogen antibody preparation can be obtained (Fig. 8). This drug has the ability to bind the extracellular domain of the Her2 / neu oncogen, as shown in the cytochemical test (Figure 9). Our estimates show that the cost of an MA preparation obtained by expression in a plant cell can be 10-20 times lower than the cost of a similar preparation obtained from animal CHO cells.

Examples

The following examples disclose the most preferred embodiments of the present invention and are provided solely for the purpose of better clarifying its essence. One skilled in the art will recognize that many modifications can be made both to the means and materials used, and to the methods used, without departing from the scope of the invention.

Example 1. Obtaining genes encoding the heavy and light chain of MA against the Her2 / neu oncogen.

From a cell culture of hybridoma ICO85 obtained in the laboratory for experimental diagnosis and biotherapy of tumors of the Research Institute of EDiTO GU RONTs im. NN Blokhin RAMS under the guidance of prof. A.Yu. Baryshnikova, sequences that encode the variable regions of the antibody affinity for the Her2 / neu oncogen were isolated by genetic engineering methods. The resulting fragments were inserted into constructs containing sequences encoding the constitutive regions of class G antibodies.

Example 2. Expression of the light and heavy chain antibodies against the Her2 / neu oncogen in a plant using a non-viral vector.

Individual light and heavy chain genes of the anti-Her2 / neu oncogene antibody were cloned into the pFF19 subclone containing the 35S promoter at the NcoI and XhoI sites. In the next step, the genes were individually cloned into the Bin 19 vector (Bevan, 1984. Nucleic Acids Res. 12, 8711-8721) at the HindIII-EcoRI sites to generate the vectors pA11860 and pA11903, respectively (Figs. 1 and 3). Agrobacterium tumefaciens with binary vectors expressing the heavy or light chain of the anti-Her2 / neu oncogen antibody was seeded in 2-YT liquid medium and grown overnight at 28 ° C. Night culture was besieged by centrifugation at 5 thousand rpm for 3 minutes The pellet was then resuspended in agroinfiltration buffer containing 10 mM MgCl ₂ and 10 mM MES pH 5.0, and introduced into the sheet in an appropriate dilution. Figure 2 shows the accumulation of the light chain (L-chain) of the anti-Her2 / neu oncogen antibody in leaves of N. benthamiana 3 days after agroinjection with the binary vector pA11866. In these experiments, we used a construct encoding the P19 anti-silencing protein of the bushy dwarf virus of tomatoes, the gene of which we synthesized earlier (Dorokhov et al., 2006. FEBS Letters 580, 3872-3878).

With the simultaneous co-injection of vectors pA11903 and pA11860 into bentamian leaves, light and heavy chains of antibodies accumulate in them, which can be isolated and purified by affinity chromatography on a protein G-Sepharose column (Figure 4).

Example 3. Expression of the light and heavy chain antibodies against the Her2 / neu oncogen in plants using viral vectors.

To create a vector based on the TMV genome (FIG. 5), the cbNA ctNA was used (Dorokhov et al. 1994, FEBS Lett. 350, 5-8), in which the replicase gene was from a closely related strain, turnip vein enlightenment virus (Lartey et al. 1994, Arch. Virol., 138, 287-298). Actin 2 promoter from Arabidopsis thaliana served as the eukaryotic transcriptional promoter (EMBL AF308778, An et al., 1996, Plant J., 10, 107-121). To create a vector based on the PVA genome (Fig.6) used the cDNA described previously (Komarova et al., 2006, Biochemistry 71, 1043-1049). Agrobacterium tumefaciens with binary vectors pA11520 and pA11333 expressing the heavy and light chains of anti-Her2 / neu oncogen antibodies, respectively, were seeded in 2-YT liquid medium and grown overnight at 28 ° C. Night culture was besieged by centrifugation at 5 thousand rpm for 3 minutes The pellet was then resuspended in agroinfiltration buffer containing 10 mM MgCl ₂ and 10 mM MES pH 5.0, and introduced into the sheet in an appropriate dilution. Fig.7 shows the accumulation of antibodies against the Her2 / neu oncogen in leaves of N. benthamiana 4 days after agroinjection with a mixture of binary vectors. You can see (Fig.7) a significant accumulation of light and heavy chains of the antibodies detected in the gel using Coomassie staining (Fig.7A) and Western analysis (Fig.7B). Using chromatography on a protein G-Sepharose column, a pure Her2 / neu anti-oncogene antibody preparation can be obtained (FIG. 8). Our experiments show that up to 300-500 mg of purified antibody can be isolated from 1 kg of leaves of N. benthamiana.

Example 4. An antibody synthesized in a plant cell is capable of binding to the Her2 / neu oncogen on the surface of SKBR-3 cancer cells.

To prove the activity of the antibodies synthesized in a plant cell, an immunocytochemical (ICC) test was used. This test, along with the radioligand and enzyme immunoassay methods, is widely used in clinical practice. Immunocytochemical analysis does not require much time, is performed quickly within 2-3 hours and is widely described in the literature (see, for example, Gluzman D.F., Sklyarenko L.M., Nadgornaya V.A., Kryachok I.A. Diagnostic immunocytochemistry of tumors. Kiev, Morion; 2003, p. 28-31). We used our antibody in the ICC of SKBR-3 cancer cells with Her2 / neu oncogen on their surface. Figure 9 shows that the antibody has the ability to bind the extracellular domain of the Her2 / neu oncogen. Thus, the antibody obtained by the method according to the present invention is fully functional and is able to specifically recognize and bind to the Her2 / neu oncogen. This opens up wide possibilities for applying the method of the present invention for the large-scale production of anti-cancer antibodies that can be used for therapeutic and diagnostic purposes. The cost of a diagnostic preparation of antibodies obtained in a plant is 12 times lower than the commercial antibodies of Dako company.

All patents, publications, scientific articles, and other documents and materials cited or referenced herein are hereby incorporated by reference to the extent that each of these documents was individually incorporated by reference or is presented in its entirety.

Specific genes, vectors, plant species, uses, materials and methods described herein are preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. When studying this description, other objects, aspects, and embodiments will come to mind to those skilled in the art, and they are encompassed by the scope of this invention. Specialists in this field will be clear that various replacements and modifications can be made in relation to the invention described here without deviating from its scope and scope, which are determined by the attached claims.

Claims (27)

1. The method of production in the plant cell of an antibody that specifically binds HER2 / neu oncoprotein, which includes
(a) providing one or more expression vectors directing in the plant cell the synthesis of the heavy, light chain of the antibody or antigen-binding fragments thereof (dsFv, scFv, scFv-Fc);
(b) introducing said one or more vectors into a plant cell;
(c) cultivating a plant cell under conditions providing for co-expression of said vectors in the cell. 2. The method according to claim 1, in which the plant cell is an isolated cell or is part of a cultured explant (tissue, organ) or as part of a whole plant. 3. The method according to claim 1, characterized in that the specified one or more expression vectors provide the synthesis of non-amplifying RNA. 4. The method according to claim 3, characterized in that the specified one or more expression vectors provide the synthesis of non-amplifying RNA using an inducible promoter. 5. The method according to claim 1, characterized in that said one or more expression vectors are viral expression vectors. 6. The method according to claim 5, characterized in that said viral expression vectors are vectors based on the genome of a DNA-containing virus. 7. The method according to claim 5, characterized in that said viral expression vectors are vectors based on the genome of an RNA-containing virus. 8. The method according to any one of claims 5 to 7, characterized in that said viral expression vectors provide for the synthesis of viral RNA using an inducible promoter. 9. The method according to claim 7, characterized in that the expression vector provides the synthesis of RNA of the tobacco mosaic virus encoding the antibody heavy chain. 10. The method according to claim 7, characterized in that the expression vector provides the synthesis of RNA of the tobacco mosaic virus encoding the antibody light chain. 11. The method according to claim 7, characterized in that the expression vector provides for the synthesis of potato X virus RNA encoding an antibody heavy chain. 12. The method according to claim 7, characterized in that the expression vector provides the synthesis of potato X virus RNA encoding the antibody light chain. 13. The method according to claim 1, characterized in that the expression vectors provide synthesis of the light and heavy chains of the antibodies in the same cell. 14. The method according to claim 5, characterized in that they use viral vectors that do not compete with each other in the same cell. 15. The method according to claim 1, characterized in that the antibody contains a signal peptide that addresses the antibody in the cell compartment or secretion outside the cell. 16. The method according to claim 1, in which the antibody belongs to an immunoglobulin class G or A, or M, or D, or E. 17. The method according to claim 1, characterized in that the plant cell is a cell of a dicotyledonous or monocotyledonous plant. 18. The method according to 17, characterized in that the dicotyledonous plant belongs to the nightshade family. 19. The method according to p. 18, characterized in that the plant in the nightshade family is a plant of the genus Nicotiana. 20. The method according to claim 19, characterized in that the plant of the genus Nicotiana is N. tabacum or N. benthamiana. 21. The method according to 17, characterized in that the dicotyledonous plant is a plant of the family Brassicacea, Legume, or Chenopodiaceae. 22. The method according to claim 1, characterized in that the vectors are introduced into the plant cell by agroinjection. 23. The method according to claim 1, characterized in that expression vectors are introduced into the cell of the plant, ensuring stable transformation. 24. The method according to claim 1, characterized in that expression vectors that provide transient transformation are introduced into the plant cell. 25. A plant cell producing an antibody that interacts with the HER2 / neu oncoprotein, transformed with one or more expression vectors directing the synthesis of the antibody heavy, light chain or its antigen-binding fragments (dsFv, scFv, scFv-Fc) in the plant cell. 26. The plant cell of claim 25, stably transformed with one or more expression vectors directing antibody synthesis. 27. The plant cell according A.25, transiently transformed with one or more expression vectors directing the synthesis of antibodies.

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